Lipoic acid analogs

ABSTRACT

A compound is provided having the formula I:  
                 
 
     wherein  
     R 1  and R 2  independently denote a methylene, ethylene or unbranched or branched C 3-16  alkylene, alkenylene or alkynylene group which is unsubstituted or substituted with one or more halogen, hydroxyl or amine groups, wherein in the unbranched or branched C 3-16  alkylene, alkenylene or alkynylene group an internal alkylene carbon atom in the carbon backbone thereof can be replaced by an oxygen atom,  
     R 3  and R 4    
     (i) independently denote  
     (a) hydrogen,  
     (b) a methyl, ethyl, vinyl or unbranched or branched C 3-16  alkyl, alkenyl or alkynyl group which is unsubstituted or substituted with one or more halogen, hydroxyl or amine groups, wherein in said unbranched or branched C 3-16  alkyl, alkenyl or alkynyl group an internal alkylene carbon atom in the carbon backbone thereof can be replaced by an oxygen atom,  
     (c) a cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl or alkynylcycloalkyl group having 15 to 16 carbon atoms which is unsubstituted or substituted with one or more halogen, hydroxyl or amine groups, or  
     (d) an aryl, alkaryl, aralkyl, alkenylaryl, aralkenyl, alkynylaryl or aralkynyl group having 6 to 16 carbon atoms which is unsubstituted or substituted with one or more halogen, hydroxyl or amine groups, or  
     (ii) jointly with the nitrogen atom form a cyclic or aromatic amine which is unsubstituted or substituted with one or more alkyl, alkenyl, alkynyl, halogen, hydroxyl or amine groups,  
     X denotes O, S, —NH— or —NR 5 —, and  
     R 5  denotes methyl, ethyl, or unbranched or branched C 3-16  alkyl,  
     or a pharmaceutically acceptable salt thereof, wherein the compound is in equilibrium at physiological pH with a protonated form thereof.  
     Methods of making the compounds, compositions including the compounds and methods of treatment of conditions involving reactive oxygen species are also provided.

FIELD OF THE INVENTION

[0001] The present invention relates to analogs of lipoic acid, inparticular lipoic acid analogs that are positively charged.

BACKGROUND OF THE INVENTION

[0002] Throughout the evolution of life on Earth, the level of oxygen inthe atmosphere increased. As a result, aerobic organisms had to developefficient defense mechanisms in order to cope with increasing oxidativestress. The toxicity of oxygen is known to be due to the formation ofreactive oxygen species (ROS) during the normal metabolism of a livingorganism. ROS include oxygen-derived free radicals and non-radicalderivatives that are capable of inciting oxidative damage to biologicalstructures. ROS have also been shown to be involved in more than onehundred different pathological syndromes and in the aging process.

[0003] There are several lines of defense against oxidative stress,including (i) macromolecules, such as enzymes, that can interact withROS directly and remove them, or chelate metals and prevent theaugmentation of oxidative damage; (ii) low molecular weight antioxidantsthat can interact directly with ROS, including both syntheticantioxidants and antioxidants from natural sources; and (iii) damagerepair mechanisms.

[0004] In the last five years, in addition to the conventional idea thatantioxidants interact with oxidants to minimize oxidative damage, a newand exciting role of these reduction-oxidation (redox) sensitivemolecules (both oxidants and anxioxidants) has become clear. Thesemolecules function in a ubiquitous redox regulation of key biologicalprocesses such as immune response, cell-cell adhesion (e.g.,atherosclerosis), cell proliferation, inflammation, metabolism, glucoseuptake (diabetes) and programmed cell death (apoptosis). The basicregulation mechanism is due to the existence of redox-regulated aminoacids in proteins, e.g., cysteine, tyrosine and methionine.Modifications of these amino acids by either oxidants or reducing agentscan trigger or inhibit the biological function of a protein.

[0005] One of the most potent naturally occurring antioxidants known islipoic acid (LA). α-Lipoic acid is also known as thioctic acid,1,2-dithiolane-3-pentanoic acid, 1,2-dithiolane-3valeric acid or6,8-thioctic acid. α-Lipoic acid has a chiral carbon atom and occurs intwo enantiomeric forms.

[0006] Biologically, lipoate exists as lipoamide in at least fiveproteins where it is covalently linked to a lysyl residue. Four of theseproteins are found in α-ketoacid dehydrogenase complexes, the pyruvatedehydrogenase complex, the branched chain keto-acid dehydrogenasecomplex and the α-ketoglutarate dehydrogenase complex. Threelipoamide-containing proteins are present in the E2 enzyme dihydrolipoylacyltransferase, which is different in each of the complexes andspecific for the substrate of the complex. One lipoyl residue is foundin protein X, which is the same in each complex. The fifty lipoamideresidue is present in the glycine cleavage system.

[0007] Recently lipoic acid has been detected in the form oflipoyllysine in various natural sources. In the plant material studied,lipoyllysine content was highest in spinach (3.15 μg/g dry weight; 92.51μg/mg protein). When expressed as weight per dry weight of lyophilizedvegetables, the abundance of naturally existing lipoate in spinach wasover three- and five-fold higher than that in broccoli and tomato,respectively. Lower concentrations of lipoyllysine were also detected ingarden pea, Brussels sprouts and rice bran. Lipoyllysine concentrationwas below detection limits in acetone powders of banana, orange peel,soybean and horseradish, however.

[0008] In animal tissues, the abundance of lipoyllysine in bovineacetone powders can be represented in the following order:kidney>heart>liver>spleen>brain>pancreas>lung. The concentration oflipoyllysine in bovine kidney and heart were 2.64±1.23 and 1.51±0.75μg/g dry weight, respectively.

[0009] Lipoate in its reduced form as dihydrolipoate (DHLA) possessestwo —SH groups which provide a very low oxidation potential to themolecule (−0.29 V). Thus, lipoic acid and the DHLA redox couple areexcellent antioxidants capable of interacting with almost all forms ofROS, recycling other antioxidants and in addition reducing oxidizeddisulfide groups in biological systems. These molecules then mayrecuperate their biological reducing power and function. All of thesequalities of LA make it also one of the most important molecules inredox signaling. A good example of this is the ability of thismetabolically active compound to increase glucose uptake in an insulinmimic effect.

[0010] Various of the enantiomeric forms of α-lipoic acid, andcombinations and derivatives thereof (including its reduced form), havebeen used to treat numerous conditions. For example, U.S. Pat. Nos.5,650,429 and 5,532,269 disclose the use of lipoic acids in thetreatment of circulatory disorders. U.S. Pat. No. 5,621,117 teaches thatthe D- and L-enantiomers of α-lipoic acid have different properties,with the D-enantiomer being primarily antiphlogistic and theL-enantiomer being mainly antinociceptive (analgesic). U.S. Pat. No.5,669,670 discloses combinations of lipoic acids and vitamins incompositions useful for producing analgesic, anti-inflammatory,antinecrotic, anti-diabetic and other therapeutic effects. U.S. Pat. No.5,334,612 describes certain alkylated derivatives of lipoic acid andtheir use in treatment of retroviral diseases. U.S. Pat. No. 5,084,481discloses the use of reduced lipoic acid (DHLA) and salts thereof intreating inflammatory diseases. U.S. Pat. No. 5,693,664 discloses use ofLA and DHLA in the treatment of diabetes. U.S. Pat. No. 5,508,275discloses a variety of lipid-selective antioxidants, including lipoicacid derivatives.

[0011] ROS are also known to be capable of activating NF-kappa B, and itis believed that ROS are the final common signal for a number of stimulithat activate NF-kappa B. Sen and Packer, The FASEB Journal, Vol. 10,709-720 (1996). The activation of NF-kappa B is believed to be involved,at least in part, in the causation or progression of a number of diseasestates. Packer et al., Advances in Pharmacology, Vol. 38, 79-101 (1997).The administration of antioxidant compositions including tocotrienyllipoates has been proposed for regulating NF-kappa B activation in U.S.Provisional Patent Application Serial No. 60/055,433, filed Aug. 4,1997, which is incorporated herein in its entirety by reference.

[0012] Lipoic acid suffers from certain disadvantages, however. Inparticular, LA is reduced to DHLA within cells and then rapidlyeffluxed.

[0013] A need exists for an improved lipoic acid analog.

[0014] In addition to reactive oxygen species (ROS), reactive nitrogenspecies (RNS) such as nitrogen monoxide and byproducts thereof, inparticular free radical byproducts thereof, have been implicated ininflammatory conditions such as diabetic neuropathy. Compositions whichinclude lipoate derivatives, in particular tocotrienyl lipoates, havealso been proposed for use in treating conditions in which RNS areinvolved, for example in U.S. Provisional Application Serial No.60/055,433.

[0015] A need exists for improved compounds that are effective intreating conditions in which RNS are involved.

SUMMARY OF THE PREFERRED EMBODIMENTS

[0016] In accordance with one aspect of the present invention, there isprovided a compound having the formula I:

[0017] wherein

[0018] R¹ and R² independently denote a methylene, ethylene orunbranched or branched C₃₋₁₆ alkylene, alkenylene or alkynylene groupwhich is unsubstituted or substituted with one or more halogen, hydroxylor amine groups, wherein in said unbranched or branched C₃₋₁₆ alkylene,alkenylene or alkynylene group an internal alkylene carbon atom in thecarbon backbone thereof can be replaced by an oxygen atom,

[0019] R³ and R⁴

[0020] (i) independently denote

[0021] (a) hydrogen,

[0022] (b) a methyl, ethyl, vinyl or unbranched or branched C₃₋₁₆ alkyl,alkenyl or alkynyl group which is unsubstituted or substituted with oneor more halogen, hydroxyl or amine groups, wherein in said unbranched orbranched C₃₋₁₆ alkyl, alkenyl or alkynyl group an internal alkylenecarbon atom in the carbon backbone thereof can be replaced by an oxygenatom,

[0023] (c) a cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl oralkynylcycloalkyl group having 5 to 16 carbon atoms which isunsubstituted or substituted with one or more halogen, hydroxyl or aminegroups, or

[0024] (d) an aryl, alkaryl, aralkyl, alkenylaryl, aralkenyl,alkynylaryl or aralkynyl group having 6 to 16 carbon atoms which isunsubstituted or substituted with one or more halogen, hydroxyl or aminegroups, or

[0025] (ii) jointly with the nitrogen atom form a cyclic or aromaticamine which is unsubstituted or substituted with one or more alkyl,alkenyl, alkynyl, halogen, hydroxyl or amine groups,

[0026] X denotes O, S, —NH— or —NR⁵—, and

[0027] R⁵ denotes methyl, ethyl, or unbranched or branched C₃₋₁₆ alkyl,

[0028] or a pharmaceutically acceptable salt thereof, wherein saidcompound is in equilibrium with a protonated form thereof.

[0029] In formula I, both the R and the S enantiomer are provided.

[0030] In a preferred embodiment, the compound is in equilibrium with aprotonated form thereof at physiological pH.

[0031] In a preferred embodiment, X denotes —NH—. More particularly, R¹is —(CH₂)₄—, R² is —(CH₂)₂—, and R³ and R⁴ are methyl groups or hydrogenatoms. The former particularly preferred embodiment is referred toherein as “LA-PLUS”.

[0032] In another preferred embodiment, X denotes —NH—, R¹ is —(CH₂)₄—,R² is —(CH₂)₂—, and R³ and R⁴ form an unsubstituted or substitutedpyridine or imidazole group together with the nitrogen atom.

[0033] In accordance with another aspect of the present invention, thereis provided a compound having the formula II:

[0034] wherein

[0035] L¹ and L² independently denote (i) a methylene group or a C₆₋₁₀arylene group which is unsubstituted or substituted with a halogen,hydroxyl, amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group or (ii) a linking group having a carbon backbone thatincludes 2 to 16 carbon atoms, wherein a carbon atom in said carbonbackbone can be replaced by an oxygen atom, an unsubstituted orsubstituted amine group, a sulfur atom, an unsubstituted or substitutedC₆₋₁₀ aryl group or a combination thereof,

[0036] Y denotes an ester, thioester, urethane or unsubstituted oralkyl-substituted amide linkage, and

[0037] A denotes a group containing a nitrogen atom that is inequilibrium with a protonated form thereof,

[0038] or a pharmaceutically acceptable salt thereof. Both the R and theS enantiomers are provided.

[0039] In accordance with a further aspect of the present invention,there is provided a compound having the formula III:

[0040] wherein

[0041] W¹, W² (i) jointly denote —S—S—, —S(O)—S— or —S—S(O)—, or (ii)individually denote a group including an oxidized sulfur atom,

[0042] L³ denotes (i) a single bond, (ii) a methylene group or a C₆₋₁₀arylene group which is unsubstituted or substituted with a halogen,hydroxyl, amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group or (iii) a linking group having a carbon backbone thatincludes 2 to 16 carbon atoms, wherein a carbon atom in said carbonbackbone can be replaced by an oxygen atom, an unsubstituted orsubstituted amine group, a sulfur atom, an unsubstituted or substitutedC₆₋₁₀ aryl group or a combination thereof,

[0043] Y denotes an ester, thioester, urethane or unsubstituted oralkyl-substituted amide linkage,

[0044] n denotes 0 or 1, and

[0045] Z denotes a group containing a nitrogen atom that is inequilibrium with a protonated form thereof and increases theintracellular retention time of said compound with respect to theanalogous compound having a carboxyl group,

[0046] or a pharmaceutically acceptable salt thereof. Both the R and theS enantiomers are provided.

[0047] In accordance with another aspect of the present invention,methods of making the inventive compounds are provided. In a firstembodiment, a method of making a compound having the formula I includesthe step of reacting a compound having the formula IV

[0048] with a compound having the formula V

R⁵—R²—NR³R⁴   V

[0049] wherein

[0050] R⁵ denotes OH, SH, NH₂ or NHR⁵.

[0051] In a second embodiment, a method of making a compound having theformula II includes the step of reacting a compound having the formulaVI

[0052] with a compound having the formula VII

Y²-L²-A   VII

[0053] wherein

[0054] Y¹ and Y² denote groups which react to form an ester, thioester,urethane or unsubstituted or substituted amide linkage.

[0055] In a third embodiment, a method of making a compound having theformula III includes the step of reacting a compound having the formulaVI

[0056] with a compound having the formula VIII

Y⁴   VIII

[0057] to form a product,

[0058] wherein

[0059] Y³ denotes Y¹ or a group that reacts with Y⁴ to form an iminegroup or a guanidine group,

[0060] Y⁴ denotes Y²-Z or a reagent that reacts with Y³ to form an iminegroup or a guanidine group, and

[0061] Y¹ and Y² denote groups which react to form an ester, thioester,urethane or unsubstituted or substituted amide linkage,

[0062] and then optionally oxidizing the product so formed.

[0063] In accordance with still another aspect of the present invention,compositions including the inventive compounds are provided. Theinventive compositions include pharmaceutical compositions as well ascosmetic preparations.

[0064] In accordance with a further aspect of the present invention,methods of treating conditions in a warm-blooded animal that involve areactive oxygen species or a redox mechanism are provided. The inventivemethods include the step of administering to a warm-blooded animalhaving the condition an effective amount of a compound as describedherein.

[0065] In preferred embodiments, the condition to be treated is apathological condition such as diabetes, atherosclerosis, an autoimmunedisease, a degenerative brain disorder such as Alzheimer's Disease,Parkinson's Disease, Huntington's Disease or epilepsy, a neoplasticdisease including cancer, trauma resulting from injuries such as headinjuries, cerebral ischemia, hepatic disorders, or AIDS. In anotherpreferred embodiment, the condition is a clinical condition in whichapoptosis or necrosis is implicated in pathogenesis.

[0066] In accordance with still another aspect of the present invention,methods of treating conditions in a warm-blooded animal that involve areactive nitrogen species are provided. The inventive methods includethe step of administering to a warm-blooded animal having he conditionan effective amount of a compound as described herein.

[0067] Other objects, features and advantages of the present inventionwill become apparent to those skilled in the art from the followingdetailed description. It is to be understood, however, that the detaileddescription and specific examples, while indicating preferredembodiments of the present invention, are given by way of illustrationand not limitation. Many changes and modifications within the scope ofthe present invention may be made without departing from the spiritthereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The invention may be more readily understood by referring to theaccompanying drawings in which

[0069]FIG. 1 is a plot showing the protection afforded againstglutamate-induced apoptosis in HT4 cells by lipoic acid (LA) and anembodiment of a compound of the invention (LA-PLUS);

[0070]FIG. 2 is a plot of HT4 cell viability (LDH leakage to medium)following exposure to glutamate;

[0071]FIG. 3 is a plot showing the down-regulation of PMA-induced ICAM-1expression by LA and LA-PLUS in human endothelial cells;

[0072]FIG. 4 is a plot showing glucose uptake in L6 rat muscle cellsfollowing treatment with LA and LA-PLUS for one hour;

[0073]FIG. 5 is a plot showing lipoamide dehydrogenase-catalyzedreduction of LA and LA-PLUS;

[0074] FIGS. 6A-B are plots showing detection of DHLA and DHLA-PLUS inthe cultured medium of cells that were treated with LA or LA-PLUS,respectively, with FIG. 6A pertaining to Jurkat T cells and FIG. 6Bpertaining to HT4 neuronal cells;

[0075]FIG. 7 is a plot showing entry of LA-PLUS into the cell andretention of DHLA-PLUS inside the cell;

[0076]FIG. 8 is a plot showing the protection of HT4 neuronal cellsagainst glutamate-induced cytotoxicity afforded by R—, S— and racemicLA-PLUS; and

[0077]FIG. 9 is a plot of the viability of HT4 cells following treatmentwith glutamate, showing the protection afforded by compounds andcompositions according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] The present invention provides analogs of lipoic acid and relatedcompounds that have unexpectedly superior biological antioxidantcapacity and redox signaling activity. This was achieved by a strategythat included converting the negative charge of the acid into a positivecharge using a group that includes a moiety that is in equilibrium witha protonated form thereof at physiological pH, and therefore bears apositive charge. The inventive compounds resemble the naturallyoccurring form of lipoic acid, which is a conjugate between lipoic acidand the amino acid lysine. A preferred embodiment of the inventivecompound (LA-PLUS) is the decarboxylation product of the naturallyoccurring form of lipoic acid, namely lipoyllysine (lipoyllysine itselfnot forming part of the present invention).

[0079] The inventive compounds can penetrate and diffuse into cells bycrossing biological membranes. The compounds are then enzymaticallyreduced and retained inside the cell, thereby potentiating theintracellular effects thereof.

[0080] The inventive compounds are active per se, and not simply aspro-drugs for lipoic acid. The positively charged form, which is inequilibrium at physiological pH with the free base, penetrates intocells and crosses biological barriers such as membranes. Inside thecells, the compounds are enzymatically reduced and serve as powerfulantioxidants.

[0081] A major drawback of LA biochemistry is that, after being taken upby cells, LA is reduced to DHLA. DHLA then is rapidly effluxed from thecell to the culture medium. Unlike LA, the inventive compounds areretained inside the cell and therefore have markedly enhanced potencycompared to the effect of LA. Blocking of the terminal carboxyl groupaccording to the invention is believed to prolong the life of theinventive compound in circulation. In blood plasma and human tissues theinventive compounds (such as LA-PLUS) are expected to have an extendedbiological half-life compared to the analogous acids (such as LA)because of slower beta oxidation and other metabolic processes to whichunprotected acids are subjected.

[0082] Embodiments of the inventive compounds are given according toformula I:

[0083] wherein

[0084] R¹ and R² independently denote a methylene, ethylene orunbranched or branched C₃₋₁₆ alkylene, alkenylene or alkynylene groupwhich is unsubstituted or substituted with one or more halogen, hydroxylor amine groups, wherein in said unbranched or branched C₃₋₁₆ alkylene,alkenylene or alkynylene group an internal alkylene carbon atom in thecarbon backbone thereof can be replaced by an oxygen atom,

[0085] R³ and R⁴

[0086] (i) independently denote

[0087] (a) hydrogen,

[0088] (b) a methyl, ethyl, vinyl or unbranched or branched C₃₋₁₆ alkyl,alkenyl or alkynyl group which is unsubstituted or substituted with oneor more halogen, hydroxyl or amine groups, wherein in said unbranched orbranched C₃₋₁₆ alkyl, alkenyl or alkynyl group an internal alkylenecarbon atom in the carbon backbone thereof can be replaced by an oxygenatom,

[0089] (c) a cycloalkyl, alkylcycloalkyl, alkenylcycloalkyl oralkynylcycloalkyl group having 5 to 16 carbon atoms which isunsubstituted or substituted with one or more halogen, hydroxyl or aminegroups, or

[0090] (d) an aryl, alkaryl, aralkyl, alkenylaryl, aralkenyl alkynylarylor aralkynyl group having 6 to 16 carbon atoms which is unsubstituted orsubstituted with one or more halogen, hydroxyl or amine groups, or

[0091] (ii) jointly with the nitrogen atom form a cyclic or aromaticamine which is unsubstituted or substituted with one or more alkyl,alkenyl, alkynyl, halogen, hydroxyl or amine groups,

[0092] X denotes O, S, —NH— or —NR⁵—, and

[0093] R⁵ denotes methyl, ethyl, or unbranched or branched C₃₋₁₆ alkyl,

[0094] or a pharmaceutically acceptable salt thereof, wherein saidcompound is in equilibrium with a protonated form thereof.

[0095] Both the R and the S enantiomer are considered to be within thescope of the present invention and specifically of formula I.

[0096] In a preferred embodiment, the inventive compound is inequilibrium with the protonated form thereof at physiological pH.

[0097] Exemplary R¹ and R² groups include: methylene, ethylene,trimethylene, tetramethylene, pentamethylene, hexamethylene,octamethylene, decamethylene, dodecamethylene, tetradecamethylene,hexadecamethylene; propylene; chloromethylene, methylmethylene;ethylethylene, propylethylene, butylethylene; 1,1-dimethylethylene,1,2-dimethylethylene, 1-methyl-2-ethylethylene, 1,1-diethylethylene;1-methyltrimethylene, 2-methyltrimethylene, 3-methyltrimethylene,1,1-dimethyltrimethylene, 1-methyl-1-ethyltrimethylene,1,2,3-trimethyltrimethylene; 1-methyltetramethylene,2,2-dimethyltetramethylene; 2-methylhexamethylene;4-methyloctamethylene; 2,4-dimethyldecamethylene; vinylene; 1-propylene,2-propylene, 1-methyl-1-propylene; 1-butenylene, 2-butenylene,3-butenylene, 1-methyl-1-butenylene, 2-ethyl-3-butenylene;1-pentenylene, 2-pentenylene, 4-ethyl-3-pentenylene; 1,3butadienylene,2-methyl-1,3-butadienylene; 1,3-pentadienylene, 2,4-pentadienylene;ethynylene; 1-propynylene, 2-propynylene, 2-methyl-1-propynylene;1-butynylene, 2-butynylene, 3-buten-1-ynylene; chloromethylene;1-chloroethylene, 2-fluoroethylene; 2,2-dichlorotetramethylene;1-hydroxyethylene; 2-hydroxytrimethylene, 1,2-dihydroxytrimethylene;1-chloro-3-hydroxyhexamethylene; 1-aminoethylene;2,4-diaminohexamethylene; 1-methyl-3-aminotetramethylene; and the like.

[0098] Additional exemplary R¹ and R² groups include groups in which atleast one non-terminal carbon atom in the carbon backbone of the groupis replaced by an oxygen atom. Such groups include, without limitation:—CH₂—O—CH₂—; —CH₂CH₂—O—CH₂—; —CH₂—O—CH₂CH₂—; —CH₂CH₂—O—CH₂CH₂—;—CH₂CH₂CH₂—O—CH₂CH₂—, —CH(CH₃)CH₂—O—CH₂CH₂—; —C(CH₃)₂CH₂—O—CH₂CH₂—;—CHClCH₂CH₂—O—CH₂CH₂—, —CH(OH)CH₂CH₂—O—CH₂CH₃—;—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₂—; —(CH₂CH₂—O)₃—CH₂CH₂—; and the like. Suchstructures include ether and conjugated ether units.

[0099] If desired, R¹ or R² can also include a substituent that is inequilibrium with a protonated form thereof at physiological pH. Such acompound will have a positive charge of at least +2. Preferably, theinventive compound bears at most two positive charges.

[0100] Exemplary R³ and R⁴ groups include: methyl, ethyl, propyl, butyl,pentyl, hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl; isopropyl;isobutyl, sec-butyl, tert-butyl; isopentyl, neopentyl, tert-pentyl;isohexyl; isooctyl; vinyl; allyl, 1-propenyl, isopropenyl; 1-butenyl,2-butenyl, 3-butenyl, 1,3-butadienyl, 2-methyl-3-butenyl; 2-pentenyl;ethynyl; 2-propynyl; 4pentynyl; chloromethyl; 1-chloroethyl,2-fluoroethyl; 2-chloropropyl; 1,1-difluorobutyl; 2-hydroxypropyl;1-hydroxybutyl, 2,3-dihydroxybutyl; 2-fluoro-4hydroxyhexyl;2-aminopropyl; 1,3-diaminohexyl; and the like.

[0101] Additional exemplary R³ and R⁴ groups include groups in which atleast one non-terminal (internal) alkylene carbon atom in the carbonbackbone of the group is replaced by an oxygen atom. Such groupsinclude, without limitation: —CH₂—O—CH₃; —CH₂CH₂—O—CH₃; —CH₂—O—CH₂CH₃;—CH₂CH₂—O—CH₂CH₃; —CH₂CH₂CH₂—O—CH₂CH₃, —CH(CH₃)CH₂—O—CH₂CH₃;—C(CH₃)₂CH₂—O—CH₂CH₃; —CF₂CH₂CH₂—O—CH₂CH₃, —CH(NH₂)CH₂CH₂—O—CH₂CH₃;—CH₂CH₂—O—CH₂CH₂—O—CH₂CH₃; —(CH₂CH₂—O)₃—CH₂CH₃; and the like.

[0102] Unsubstituted and substituted cyclic R³ and R⁴ groups includecyclopentyl, cyclohexyl, 4-chlorocyclohexyl, 3,5-dimethylcyclohexyl,4-tert-butylcyclohexyl, 3-cyclohaxylbutyl, 4-cyclohexyl-2-butenyl, andthe like. Polycyclic groups can also be used.

[0103] Unsubstituted and substituted aromatic R³ and R⁴ groups includephenyl, 4-chlorophenyl, 4-ethylphenyl, 3-phenylpropyl,2-chloro-5-phenylpentyl, and the like. Polycyclic aromatic groups suchas naphthyl, and partially aromatic polycyclic groups such as indyl,which can be further substituted with alkyl, alkenyl, alkynyl, halogen,hydroxyl or amine groups, can also be used.

[0104] Furthermore R³ and R⁴ can, together with the nitrogen atom towhich they are bonded, form a cyclic or aromatic amine group, includingpolycyclic groups. Examplary groups formed jointly by R³, R⁴ and N informula I include: 1-pyridyl; 3-carboxy-1-pyridyl (nicotinic acidderivative); 1-imidazolyl; 1-indolyl; 1-piperidyl; 1-pyrazolyl;1-benzpyrazolyl; and the like. The cyclic or aromatic amine groups canalso be further substituted with one or more alkyl, alkenyl, alkynyl,halogen, hydroxyl or amine groups.

[0105] As with R¹ and R², the R³ and R⁴ groups can also include anadditional substituent which is positively charged at physiological pH,resulting in a compound with a charge of at least +2. Again, it ispreferable that the total charge of the inventive compound not exceed+2. More particularly, the charge of the inventive compound is +1 atphysiological pH when it is dissociated from its counterion in solution.

[0106] Preferred embodiments include those in which X=—NH—, and morespecifically those in which R¹ and R² are polyalkylene groups, and R³and R⁴ are methyl groups or jointly form an unsubstituted or substitutedpyridine or imidazole group together with the nitrogen atom.Particularly preferred is the “LA-PLUS” embodiment in which R¹ is—(CH₂)₄—, R² is —(CH₂)₂—, and R³ and R⁴ are methyl groups. In apreferred variant of LA-PLUS, R³ and R⁴ are hydrogen atoms.

[0107] More generally, compounds according to the invention are providedaccording to formula II:

[0108] In formula II,

[0109] L¹ and L² independently denote (i) a methylene group or a C₆₋₁₀arylene group which is unsubstituted or substituted with a halogen,hydroxyl, amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group or (ii) a linking group having a carbon backbone thatincludes 2 to 16 carbon atoms, wherein a carbon atom in said carbonbackbone can be replaced by an oxygen atom, an unsubstituted orsubstituted amine group, a sulfur atom, an unsubstituted or substitutedC₆₋₁₀ aryl group or a combination thereof,

[0110] Y denotes an ester, thioester, urethane or unsubstituted oralkyl-substituted amide linkage, and

[0111] A denotes a group containing a nitrogen atom that is inequilibrium with a protonated form thereof,

[0112] or a pharmaceutically acceptable salt thereof.

[0113] In Formula II, A includes all groups of the form —NR³R⁴ as setforth above. Further exemplary A groups include imine groups having theformulas IIa-b:

—C(R⁷)=N—R⁸   IIa

—N=CR⁷R⁸   IIb

[0114] wherein

[0115] R⁷, R⁸ denote hydrogen, a methyl group, a C₆₋₁₀ aryl group whichis unsubstituted or substituted with a halogen, hydroxyl, amine orunbranched or branched C₃₋₁₆ alkyl, alkenyl or alkynyl group, or anunbranched or branched C₂₋₁₆ alkyl, alkenyl or alkynyl group which isunsubstituted or substituted with a halogen, hydroxyl or amine group,

[0116] and guanidine groups having the formula IIc:

[0117] wherein

[0118] R⁹, R¹⁰ denote hydrogen or C₁₋₆ alkyl.

[0119] The A group can also include additional linking groups, forexample additional L¹ groups, and additional ester, thioester, urethaneand/or amide linkages, affording a polymer-like structure of the form-Y-L-Y- . . .

[0120] Exemplary L¹ and L² groups include those set forth above inconnection with the R¹ and R² groups of formula I. Additional exemplaryL¹ and L² groups include groups in which at least one non-terminalcarbon atom in the C₂₋₁₆ carbon backbone of the group is replaced by asulfur atom. Such groups include, without limitation: —CH₂—S—CH₂—;—CH₂CH₂—S—CH₂—; —CH₂—S—CH₂CH₂—; —CH₂CH₂—S—CH₂CH₂—; —CH₂CH₂CH₂—S—CH₂CH₂—,—CH(CH₃)CH₂—S—CH₂CH₂—; —C(CH₃)₂CH₂—S—CH₂CH₂—; —CHClCH₂CH₂—S—CH₂CH₂—,—CH(OH)CH₂CH₂—S—CH₂CH₃—; —CH₂CH₂—S—CH₂CH₂—S—CH₂CH₂—;—(CH₂CH₂—S)₃—CH₂CH₂—; and the like.

[0121] Further exemplary R¹ and R² groups include groups in which atleast one carbon atom in the C₂₋₁₆ carbon backbone of the group isreplaced by an unsubstituted or substituted amine group. In the lattercase, the amine group preferably is substituted with an unbranched orbranched C₃₋₁₆ alkyl, alkenyl or alkynyl group. Such groups include,without limitation: —CH₂—NH—CH₂—; —CH₂—N(CH₃)—CH₂—; —CH₂—N(CH₂CH₃)—CH₂—;—CH₂CH₂—NH—CH₂—; —CH₂—N(CH₂CH₃)—CH₂CH₂—; —CH₂CH₂—NH—CH₂CH₂—;—CH₂CH₂CH₂—N(CH₂CH₂CH₃)—CH₂CH₂—; —CH(CH₃)CH₂—NH—CH₂CH₂—;—C(CH₃)₂CH₂—NH—CH₂CH₂—; —CH₂CH₂CH₂—N(CH₂CH₂CH₃)—CH₂CH₂—; and the like.

[0122] Additional L¹ and L² groups include unsubstituted or substitutedC₆₋₁₀ arylene groups, and gropus in which one or more carbon atoms inthe C₂₋₁₆ carbon backbone thereof are replaced with an unsubstituted orsubstituted aryl group. Combinations of aryl and oxygen, sulfur and/oramine groups can also be included, as well as partially aromaticpolycyclic groups. Polymeric structures including repeated aryl,alkaryl, etc. units can also be included.

[0123] Exemplary L¹ and L² groups include without limitation:

[0124] and the like.

[0125] Still more generally, the invention provides compounds accordingto the formula III:

[0126] In formula III,

[0127] W¹, W² (i) jointly denote —S—S—, —S(O)—S— or —S—S(O)—, or (ii)individually denote a group including an oxidized sulfur atom,

[0128] L³ denotes (i) a single bond, (ii) a methylene group or a C₆₋₁₀arylene group which is unsubstituted or substituted with a halogen,hydroxyl, amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group or (iii) a linking group having a carbon backbone thatincludes 2 to 16 carbon atoms, wherein a carbon atom in said carbonbackbone can be replaced by an oxygen atom, an unsubstituted orsubstituted amine group, a sulfur atom, an unsubstituted or substitutedC₆₋₁₀ aryl group or a combination thereof,

[0129] Y denotes an ester, thioester, urethane or unsubstituted orsubstituted amide linkage,

[0130] n denotes 0 or 1, and

[0131] Z denotes a group that is in equilibrium at physiological pH witha protonated form thereof and increases the intracellular retention timeof said compound with respect to the analogous compound having acarboxyl group,

[0132] or a pharmaceutically acceptable salt thereof. Both the R and theS enantiomers are provided.

[0133] As with the A group in formula II, in formula III the Z group caninclude additional L and Y groups. Compounds according to formula IIIalso include those in which n=0 (no Y linkage) and L³ is a single bond.In these compounds, the Z group is bonded directly to the dithiolanering. Exemplary Z groups include imines and guanidines, as well asgroups of the formula —NR³R⁴.

[0134] Compounds according to formula III in which W¹ and W² jointlydenote —S—S— are typically non-metabolized forms. The present inventionalso contemplates and encompasses metabolites of such compounds, inparticular, metabolites in which one or both sulfur atoms are oxidized.Such compounds include those compounds of formula III in which W¹ and W²jointly denote —S(O)—S— or —S—S(O)— (thiolsulfinates), or individuallydenote a group including an oxidized sulfur atom, such as an SO₃H group.

[0135] Methods for making the inventive compounds are also provided. Ina first method, a compound having the formula I includes the step ofreacting a compound having the formula IV

[0136] with a compound having the formula V

R⁸—R²—NR³R⁴   V

[0137] wherein

[0138] R⁵ denotes OH, SH, NH₂ or NHR⁵.

[0139] Exemplary reagents within the scope of formula IV include lipoicacid (R¹=—(CH₂)₄—).

[0140] Exemplary reagents within the scope of formula V include:ethylenediamine; N,N-dimethylethylenediamine; N,N-dimethylethanolamine;tetramethylenediamine; hexamethylenediamine; 1-pyridylethyleneamine;1-imidazolyltetramethyleneamine; and the like.

[0141] In the inventive method, the reagents are preferably employed ina ratio from about 1:1 to about 1:3 (formula IV:formula V).

[0142] In another embodiment, a method of making a compound having theformula II includes the step of reacting a compound having the formulaVI

[0143] with a compound having the formula VII

Y²-L²-A   VII

[0144] wherein

[0145] Y¹ and Y² denote groups which react to form an ester, thioester,urethane or unsubstituted or substituted amide linkage.

[0146] Exemplary combinations of Y¹ and Y² include —COOH/—NH₂;—COOH/—NHCH₃; —COOH/—OH; —COOH/—SH; —OH/—NCO; —OC(O)Cl/—NH₂; and thelike. In the foregoing, either Y¹ or Y² can be the first element of thecombination, with the remaining group being the second element.

[0147] In a further embodiment, a method of making a compound having theformula III includes the step of reacting a compound having the formulaVI

[0148] with a compound having the formula VIII

Y⁴   VIII

[0149] to form a product,

[0150] wherein

[0151] Y³ denotes Y¹ or a group that reacts with Y⁴ to form an imine orguanidine group,

[0152] Y⁴ denotes Y²-Z or a reagent that reacts with Y³ to form an imineor guanidine group, and

[0153] Y¹ and Y² denote groups which react to form an ester, thioester,urethane or unsubstituted or substituted amide linkage, and thenoptionally oxidizing the product.

[0154] Exemplary reactions according to the foregoing method include thefollowing: formation of imines (L³=a single bond), with Y³ an aldehydeor ketone group having the formula —C(O)—R⁷ and Y⁴ an amine having theformula H₂N—R⁸; formation of imines (L³=a single bond), with Y³ analdehyde or ketone having the formula C(O)R⁷R⁸ and Y⁴ an —NH₂ group;formation of guanidines (L³=a single bond), with Y³ an alkylisouroniumgroup having the formula —HN⁺=

[0155] (Alk denoting an alkyl

[0156] group, preferably a C₂₋₄ n-alkyl group) and Y⁴ an amine havingthe formula R¹⁰—NH₂. Such reactions result in formation of imine andguanidine groups Z.

[0157] Useful solvents for carrying out the reactions according to theinvention include dichloromethane (DCM), chloroform, acetonitrile,tetrahydrofuran (THF), N,N-dimethylformamide (DMF) and othernon-protonated solvents.

[0158] Useful coupling reagents for esterification include thionylchloride, oxalyl chloride, phosphorus trichloride (PCl₃), anddicyclohexylcarbodiimide (DCC). The reactions are advantageously carriedout at mild reflux temperature.

[0159] For amidation, useful coupling agents include DCC,1-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (NHS),benzotriazol-1-yloxy-tris(dimetylamino)phosphonium hexafluorophosphate(BOP), and the like. Amidation can also be carried out by forming theacyl chloride from the acid and reacting it with the appropriate aminein the presence of an excess of the amine or in the presence of anothertertiary amine. The reaction can also be carried out by heating theamine and the acid. For amidation reactions, the reaction mixturepreferably is stirred overnight at room temperature.

[0160] The inventive compounds advantageously are further purified byrecrystallization from solvents such as acetone or diethyl ether at lowtemperatures (e.g., about −20° C. to 4° C.).

[0161] Pharmaceutical compositions including the inventive compounds arealso provided. The preparation of the pharmaceutical compositions of theinvention is carried out in known manner.

[0162] Useful carriers and auxiliaries for the inventive compositionsinclude gelatin, natural sugars such as raw sugar or lactose, lecithin,pectin, starches (e.g., corn starch or amylose), cyclodextrins andcyclodextrin derivatives, dextran, polyvinyl pyrrolidone, polyvinylacetate, gum arabic, alginic acid, tylose, talcum, lycopodium, silicagel (e.g., colloidal silica gel), cellulose, cellulose derivatives suchas cellulose ethers (e.g., HPMC), C₁₂₋₂₂ fatty acids and magnesium,calcium or aluminum salts thereof (e.g., stearates), emulsifiers, oilsand fats, in particular vegetable (e.g., peanut oil, castor oil, oliveoil, sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflowerseed oil, cod liver oil), glycerol esters and partial or completepolyglycerol esters of saturated fatty acids, pharmaceuticallyacceptable mono- or multivalent alcohols and polyglycols (e.g.,polyethylene glycol and derivatives thereof), esters of C₂₋₂₂ aliphaticsaturated or unsaturated fatty acids (preferably C₁₀₋₁₈ acids) withmonovalent C₁₋₂₀ aliphatic alcohols or multivalent alcohols such asglycols, glycerol, diethylene glycol, pentaerythritol, sorbitol,mannitol and the like, which may optionally also be etherified, estersof citric acid with primary alcohols, acetic acid, urea, benzylbenzoate, dioxolanes, glyceroformals, tetrahydrofurfuryl alcohol,polyglycol ethers with C₁₋₁₂ alcohols, dimethylacetamide, lactamides,lactates, ethylcarbonates, silicones (e.g., medium-viscositypolydimethyl siloxanes), calcium carbonate, sodium carbonate, calciumphosphate, sodium phosphate, magnesium carbonate, etc.

[0163] Additional useful additives include disintegrants, such ascross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodiumcarboxymethyl cellulose or microcrystalline cellulose.

[0164] The inventive compositions can be coated with conventionalcoating materials such as polymers and copolymers of acrylic acid and/ormethacrylic acid and/or their esters (e.g., Eudragit® copolymers),polyvinyl acetate, fats, oils, waxes, fatty alcohols, hydroxypropylmethyl cellulose phthalate or -acetate succinate, cellulose acetatephthalate, starch acetate phthalate, polyvinyl acetate phthalate,carboxy methyl cellulose, methyl cellulose phthalate, methyl cellulosesuccinate, zein, ethyl cellulose, ethyl cellulose succinate, shellac,gluten, ethylcarboxyethyl cellulose, ethacrylate-maleic acid anhydridecopolymer, maleic acid anhydride-vinyl methyl ether copolymer,styrol-maleic acid copolymerizate, 2-ethyl-hexyl-acrylate maleic acidanhydride, crotonic acid-vinyl acetate copolymer, glutaminicacid/glutamic acid ester copolymer, carboxymethylethylcellulose glycerolmonooctanoate, cellulose acetate succinate, and polyarginin.

[0165] Useful plasticizing agents that can be used as coating materialsinclude citric and tartaric acid esters (acetyltriethyl citrate, acetyltributyl-, tributyl-, triethyl-citrate), glycerol and glycerol esters(glycerol diacetate, -triacetate, acetylated monoglycerides, castoroil), phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-,dipropyl-phthalate), di-(2-methoxy- or 2-ethoxyethyl)-phthalate,ethylphthalyl glycolate, butylphthalylethyl glycolate andbutylglycolate; alcohols (propylene glycol, polyethylene glycol ofvarious chain lengths), adipates (diethyladipate, di-(2-methoxy- or2-ethoxyethyl)-adipate, benzophenone; diethyl- and dibutylsebacate,dibutylsuccinate, dibutyltartrate, diethylene glycol dipropionate,ethyleneglycol diacetate, -dibutyrate, -dipropionate, tributylphosphate, tributyrin, polyethylene glycol sorbitan monooleate(polysorbates such as Polysorbat 80), and sorbitan monooleate.

[0166] When the inventive compositions are solutions or suspensions, itis useful to employ solvents such as water or physiologically acceptableorganic solvents including alcohols such as ethanol, propanol,isopropanol, 1,2-propylene glycol, polyglycols and their derivatives,fatty alcohols, partial esters of glycerol, oils such as peanut oil,olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castoroil, bovine hoof oil, paraffins, dimethyl sulphoxide, triglycerides andthe like.

[0167] Injectable solutions or suspensions including the inventivecompounds can be prepared using non-toxic parenterally acceptablediluting agents or solvents, such as water, 1,3-butanediol, ethanol,1,2-propylene glycol, polyglycols mixed with water, glycerol, Ringer'ssolution, isotonic salt solution or also hardened oils includingsynthetic mono- or diglycerides or fatty acids such as oleic acid.

[0168] Solubilizers and emulsifiers useful in preparing compositionsaccording to the invention include polyvinyl pyrrolidone, sorbitan fattyacid esters such as sorbitan trioleate, phosphatides such as lecithin,acacia, tragacanth, polyoxyethylated sorbitan monooleate and otherethoxylated fatty acid esters of sorbitan, polyoxyethylated fats,polyoxyethylated oleotriglycerides, linolizated oleotriglycerides,polyethylene oxide condensation products of fatty alcohols, alkylphenolsand fatty acids.

[0169] For aqueous injection and drinkable solutions, the followingstabilizers and solubilizers can be advantageously employed: C₂₋₄aliphatic mono- and multivalent alcohols such as ethanol, n-propanol andglycerol, polyethylene glycols with molecular weights between 200-600,diethylene glycol monoethyl ether, 1,2-propylene glycol, organic amides(e.g., amides of aliphatic C₁₋₆ carboxylic acids with ammonia orprimary, secondary or tertiary C₁₋₆ amines or C₁₋₄ hydroxy amines suchas urea, urethane, acetamide, N-methyl acetamide, N,N-diethyl acetamide,N,N-dimethyl acetamide, C₂₋₆ aliphatic amines and diamines such asethylene diamine, hydroxyethyl theophylline, tromethamine (e.g., as 0.1to 20% aqueous solution), and aliphatic amino acids.

[0170] Preservatives, stabilizers, buffer substances, flavor correctingagents, sweeteners, colorants and antioxidants can also be employed.Antioxidants can be employed as preservatives, but at higherconcentrations antioxidants may serve as co-active ingredients with theinventive compounds (i.e., synergistic activity may occur).

[0171] Useful antioxidants include sodium sulphite, sodium hydrogensulphite, sodium metabisulphite, selenium, inorganic and organicselenium compounds and salts such as sodium selenite, ascorbic acid,ascorbyl palmitate, myristate and stearate, gallic acid, gallic acidallyl ester, butylhydroxyanisol, nordihydroguaiacic acid andtocopherols, as well as synergisms (substances which bind heavy metalsthrough complex formation, for example lecithin, ascorbic acid,phosphoric acid ethylene diamine tetraacetic acid, citrates, tartrates).Compositions including selenium salts such as sodium selenite haveproven particularly beneficial.

[0172] Preservatives useful in the inventive compositions include, forexample, sorbic acid, p-hydroxybenzoic acid esters, benzoic acid, sodiumbenzoate, trichloroisobutyl alcohol, phenol, cresol, benzethoniumchloride, chlorhexidine and formalin derivatives.

[0173] The pharmaceutical compositions may be applied to the skin ormucous membranes or to the inside of the body, application for examplebeing oral, enteral, pulmonal, nasal, lingual, intravenous,intra-arterial, intracardial, intramuscular, intraperitoneal,intracutaneous, subcutaneous and transdermal.

[0174] The inventive compounds can also be formulated as cosmeticpreparations, such as lotions, sun screens, ointments, solutions,creams, liposomes and emulsions. Conventional cosmetic additives,carriers, etc. can be combined with the inventive compounds to producethe desired cosmetic preparations in accordance with methods known tothose skilled in the art.

[0175] The compounds of the present invention are more potent thanlipoic acid in preventing expression of adhesion molecules on themembrane surface of endothelial cells, in increasing glucose uptake inrat muscle cells, and in protecting cells against reactive oxygenspecies and apoptosis (programmed cell death). The inventive compoundsare useful in treating a variety of conditions that involve reactiveoxygen species, redox mechanisms and reactive nitrogen species,including without limitation pathological conditions such asexercise-induced tissue damage and physical performance, diabetes (typeI and type II), atherosclerosis, an autoimmune disease, a degenerativebrain disorder such as Alzheimer's Disease, Parkinson's Disease,Huntington's Disease, epilepsy, a neoplastic disease including cancer,trauma resulting from injuries such as head injuries, ischemic andreperfusion injuries (e.g., cerebral stroke), hepatic disorders such ashepatic biliary cirrhosis, or AIDS, as well as clinical conditions inwhich apoptosis or necrosis are implicated in pathogenesis. Otherconditions which are beneficially treated by the inventive compoundsinclude muscle wasting (cachexia) in cancer and AIDS. The inventivecompounds are also beneficially employed in slowing the aging process bycombating the intrinsic oxidations that cause injury throughout life.

[0176] Tablet and capsule compositions of the invention advantageouslyinclude up to about 1000 mg of the inventive compound. Emulsions andliposomes advantageously include up to about 10% (w/v) of the inventivecompound. Micellar solutions beneficially contain up to about 25 wt %(w/v) of the inventive compound. Combinations of two or more of theinventive compounds can also be employed in the inventive compositions.

[0177] The exact dosage used will vary depending on the particularcompound of the invention selected, the age, size, and health of thesubject, and the nature and severity of the condition to be treated.However, the appropriate dosage may be determined by one of ordinaryskill by routine experimentation, following the guidance set forthherein. As a general guideline, a compound of the invention may beadministered daily in a dose of up to about 25 mg/Kg body weight (orabout 1.5 g for a 70 Kg subject) as a dietary supplement for prophylaxisfrom diseases due to oxidative stress. One may administer up to about 50mg/Kg (or about 3 g) per day for treatment of acute oxidative stress.Suitable subjects include, without limitation, mammals, such as humans,horses, cattle, swine, dogs, cats, and the like, and cells in culture,including mammalian cells, yeast, bacterial cells and the like.

[0178] The present invention is illustrated by the followingnon-limiting examples.

EXAMPLE 1 Synthesis of N,N-Dimethyl-N-2-Amidoethyl Lipoate (HCl)(“LA-PLUS”)

[0179] One (1) equivalent of lipoic acid was dissolved in 100 mlanhydrous dichloromethane (DCM). Three equivalents ofN,N-dimethylethylendiamine were then added, and the mixture was stirredfor 5 min. Next, 1.2 equivalents of N-hydroxysuccinimide, dissolved in 5ml acetonitrile, were added, followed by 1.2 equivalents ofdicyclohexylcarbodiimide (DCC) dissolved in 5 mL DCM. The mixture waskept at room temperature overnight under stirring. Then the reactionmixture was transferred into a separatory funnel. The compound wasextracted into the aqueous phase by adding concentrated HCl up toacidified pH, and then was extracted back to a 100 ml portion ofchloroform using 1 N NaOH solution. The organic layer was separated fromthe aqueous layer and dried with anhydrous magnesium sulfate, filteredand evaporated to dryness.

[0180] The compound was then re-dissolved in dichloromethane. HCl gaswas bubbled into the organic solvent up to saturation. The DCM wasevaporated and the HCl salt of N,N-Dimethy,N-2-Amidoethyl Lipoate(LA-PLUS) was precipitated by using anhydrous ether. Yield 80%.

[0181] The following tests were conducted using LA and LA-PLUS:

[0182] Glutamate Cytotoxicity: Cell Culture

[0183] HT-4 cells (mouse hippocampal cell line) were purchased fromAmerican Type Culture Collection (ATCC, Bethesda, Md.). All experimentsreported here were carried out with 10 mM glutamate.

[0184] HT4 cells were grown in DMEM supplemented with 10% serum and 100U/ml penicillin-streptomycin at 37° C. in a humidified atmospherecontaining 95% air/5% CO₂.

[0185] All experiments were performed under the following protocol.Cells were allowed to grow to confluency, were trypsinized andsubcultured in culture dishes at a concentration of 375,000 cells permillititer. The cells were continually cultured at 37° C. and wereallowed to grow for 24 h, and then were exposed to glutamate treatment.

[0186] Determination of Cell Viability

[0187] Cell viability was assayed using lactate dehydrogenase (LDH)leakage. After completion of the experiments, culture medium was removedfrom plates and centrifuged (500 g×5 min). The cells that detached fromthe monolayer following glutamate treatment were separated bycentrifugation of the growth medium. The supernatant medium was mixedwith an equal volume of bovine serum albumin (BSA) solution (5% in PBS)to help stabilize LDH activity in the solution saved for storage at 4°C. The pelleted detached cells were washed once with PBS and treatedwith a lysis buffer (Triton X-100 0.5% v/v in PBS). The resulting lysatewas mixed with an equal volume of the BSA solution for storage at 4° C.Attached cells were washed with PBS, treated with the lysis buffer withBSA solution as described above for detached cells. LDH activity wasmeasured from the samples spectophotometrically within two days ofstorage. Cell viability was determined using the following formula:viability=attached cell LDH activity/total LDH activity (medium+detachedcell LDH activity+attached cell LDH activity).

[0188] Flow Cytometric Assay of Fragmented DNA

[0189] HT4 cells were exposed to 10 mM glutamate for 24 h with andwithout antioxidants. Following the treatment the cells weretrypsinized, centrifuged, and the pellet was resuspended in a solutioncontaining propidium iodide (50 μg/ml), sodium citrate (0.1%) andTriton-X 100 (0.1%). The permeablized cells were kept in the dark for 6h at 4° C. and their DNA integrity was analyzed using a flow cytometer(XL, Coulter, FL) with argon-ion laser (excitation 488 nm) and the 575nm emission was recorded in fluorescence channel 1 (FL1). Data werecollected from 40,000 cells. The ratio of fragmented/intact DNArepresent the measure of apoptosis.

[0190] Enzymatic Reduction of LA and LA-PLUS by LipoylamideDegydrogenase

[0191] A reaction mixture was prepared including 0.1 mM of NADH, 0.1U/ml of the enzyme and 0.3 mM of LA or LA-PLUS at room temperature. Theconversion rate of LA to DHLA or LA-PLUS to DHLA-PLUS (the reduced formof LA-PLUS, analogous to DHLA) was measured by following the oxidationrate of NADH spectrophotometrically at 340 nm.

[0192] Assay for Cells Ability to Retain the Reduced Forms of LA orLA-PLUS

[0193] Jurkat T cells or neuronal HT4 cells were incubated with LA (0.1mM) or LA-PLUS (0.1 mM). The medium of the cells was replaced byphosphate buffer saline that contained 250 uM of5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The free thiols in themedium were trapped by the DTNB and absorption was measured at 420 nm atdifferent time intervals.

[0194] Adhesion Molecules Expression

[0195] Cells and Cell Culture

[0196] EV304 (ECV), a spontaneously transformed immortal humanendothelial cell line established from the vein of an apparently normalhuman umbilical cord, were obtained from ATCC (Bethesda, Md.). ECV cellswere grown in medium 199 supplemented with 10% fetal calf serum, 100U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in astandard culture incubator with humidified air containing 5% CO2 at 37°C.

[0197] Expression of Adhesion Molecules

[0198] ECV monolayers were pretreated with LA or LA-PLUS for the timeperiod indicated in the respective figure legend. Following thepretreatment, cells were activated using 20 nM PMA for 24 h. To studyICAM-1 expression, cells were incubated with fluorescein isothiocyanate(FITC) coupled to ICAM monoclonal antibody (Immunotech, Cedex, France)for 30 min at 4° C. Cells were washed twice in Dulbecco's PBS, pH 7.4(DPBS) and finally resuspended in fresh D-PBS. Expression of ICAM-1 wasimmediately assayed with a flow cytometer as described in the followingsection. Appropriate isotypic controls were used for backgroundfluorescence in the ICAM-1 assay.

[0199] The fluorescence and light scattering properties (forwardscatter, FS; side scatter, SS) of the cells were determined by using anEPICS-Elite (Coulter, Miami, Fla.) flow cytometer. Cells withFITC-conjugated antibodies were excited using a 488 nm argon ion laserand emission of FTIC was recorded at 525 nm. In each sample, at least10,000 gated viable cells were examined. A logarithmic scale was used tomeasure both background and endothelial cell fluorescence. Backgroundfluorescence was then subtracted from endothelial cell fluorescence,allowing linear comparisons of ICAM-1 expression between varioussamples.

[0200] Glucose Uptake by L6 Rat Muscle Derived Cells

[0201] L6 cells were obtained from American Type Culture Collection(MD). Dulbecco's Phosphate Buffered Saline (DPBS) and Dulbecco'sModified Eagle Medium (DMEM) supplemented with high glucose,L-glutamine, pyridoxine hydrochloride and 110 mg/l sodium pyruvate wereobtained from GIBCO BRL (Life Technologies NY). Fetal calf serum (FCS)and other reagents for the culture medium were obtained from the cellculture facility of the University of California-San Francisco.

[0202] Cell Culture

[0203] Undifferentiated mononucleated cells (myoblasts) were used inthis study. For experiments, cells were seeded at a concentration of0.2×10⁶ cells per well in 6-well flat bottom tissue culture treatedpolystyrene plates (FALCON, Becton Dickinson Labware, NJ). Cultures weregrown in DMEM supplemented with 10% FCS, 5 mM glutamine, 0.3% D-glucose,50 U/ml of penicillin, and 50 mg/ml of streptomycin, in humidified aircontaining 5% CO₂ at 37° C. Cell viability was greater than 97% asestimated by trypan blue exclusion or propidium iodide staining. Thecells were split once every third day. All experiments were carried outon cells (6th to 12th passage) in the third day of the last passage.

[0204] Cells seeded in 6-well plates were washed thrice with 1 mltransport suffer (140 mM NaCl, 5 mM KCl, 2.5 mM MgCl₂, 1 mM CaCl₂ in 20mN tris-HEPES, pH 7.4) at room temperature. Following washing, 1 ml offresh transport buffer was added to the monolayers, at room temperature.LA or LA PC were added to the cells. Cytochalasin B (finalconcentration: 20 μM) was added to the specific wells to determine thenon-specific binding of [¹⁴C(U)]-deoxy-D-glucose (DG) to the cells. The[¹⁴C(U)]-DG was added to the cells for the uptake experiments following1 h of LA or LA-PLUS treatments. The uptake of [¹⁴C(U)]-DG was carriedout for 10 min. The reaction was stopped by adding 3 ml of ice-coldtransfer buffer to each well using a dispenser. Each well was washedthrice with ice-cold transport buffer followed by lysis of the cellsusing 0.5 ml of 10% SDS. Radioactivity in each sample was read using ascintillation counter.

[0205] Results

[0206] To evaluate whether glutamate treatment induced apoptosis in theHT4 cells or facilitated cell death by a necrosis mechanism, the cellswere treated for 24 hours with glutamate. The pathway of cell death wasexplored by staining the cells for 6 h following the glutamate treatmentwith a DNA-binding fluorescent dye, propidum iodide (PI) in cellspermeabilised with a low concentration of detergent (Triton X-100 0.1%w/v). The dye which penetrated to the permeabelized cells stained thenuclear DNA. The cells were analyzed by flow cytometry.

[0207] Results showed glutamate-induced cell shrinkage and DNAfragmentation—two characteristic features of apoptosis. In the presenceof 50 μM of LA-PLUS the apoptotic process was completely blocked. Theanti-apoptotic effect of LA-PLUS in this model was much more pronouncedcompared to the effect of LA (FIG. 1).

[0208] The viability of cells following glutamate treatment was checkedin the presence or absence of LA and LA-PLUS. LA-PLUS was found to bemore efficient in protecting the HT4 cells than lipoic acid, 50 μM ofLA-PLUS were sufficient to completely protect the cells againstglutamate cytotoxicity, while LA even at 100 μM did not provide totalprotection (FIG. 2).

[0209] Expression of the adhesion molecule ICAM-1 was induced in humanendothelial ECV cells by phorbol ester (phorbol myristate acetate, PMA).PMA induced ICAM-1 expression in the cells was much more effectivelydown-regulated by LA-PLUS compared to LA when compared for the sameconcentration used.

[0210] Stimulation of glucose uptake by L6 myoblasts was studied inresponse to pretreatment of the cells with LA or LA-PLUS for 1 h. FIG. 4shows that LA-PLUS was clearly more effective than LA in enhancingglucose uptake by the muscle derived cells.

[0211] In order to check whether LA-PLUS is a biologically activemetabolic antioxidant the ability of the most specific enzyme thatutilized lipoic acid as a substrate, lipoylamide dehydrogenase, toreduce oxidized LA or LA-PLUS to their reduced forms was tested. Theconversion of lipoic acid (LA) to dihydrolipoate (DHLA) or LA-PLUS toDHLA-PLUS by purified lipoylamide dehydrogenase (0.1 U/mL) was followedby monitoring NADH consumption at 340 nm. The enzyme was able to reducethe R-stereoisomer of LA-PLUS 30 times faster than the correspondingform of LA (FIG. 5).

[0212]FIG. 6 describes the basic difference in the mechanism of actionof LA and LA-PLUS.

[0213] The efflux of DHLA and DHLA-PLUS from cells was measured in themedium of cells in culture. The medium was composed of PBS and DTNB.Release of DHLA and DHLA-PLUS from Jurkat T cells (FIG. 6A) and HT4neuronal cells (FIG. 6B) resulted in production of yellow color (TNB),which was measured spectrophotometrically at 420 nm. The results showthat the reduced form of LA is effluxed from the cell faster thanDHLA-PLUS, which is retained inside the cells (FIG. 7). An illustrationof how LA-PLUS crosses biological barriers such as membranes (alsoshowing retention of DHLA-PLUS inside the cell) is given in FIG. 7.

[0214] Three different stereo-isomers of LA-PLUS were prepared in orderto evaluate whether the protection against glutamate-induced apoptosisin neuronal cells, and the antioxidant activity of the compound, werestereospecific. The ability of (R)-LA-PLUS, (R)(S)-LA-PLUS, or(S)-LA-PLUS (50 μM) to protect HT4 cells against glutamate-inducedapoptosis was measured. All three compounds were found to be effectiveinhibitors of the apoptotic cascade (FIG. 8).

EXAMPLE 2 Synthesis of N,N-Dimethylaminoethyl Lipoate (HCl) (“LA-PLUSEster”)

[0215] One (1) equivalent of lipoic acid was dissolved in 100 mlanhydrous DCM. Two equivalents of N,N-dimethylethanolamine were added tothe mixture. Five equivalents of thionyl chloride were added dropwise.The mixture was stirred for 30 min, then refluxed for 2 h. The organicsolvent was then removed by reduced pressure evaporation, and the esterwas crystallized at −20° C. from diethyl ether.

EXAMPLE 3 Emulson Composition

[0216] The following composition is prepared: LA-PLUS 1 g triglycerideoil 10 g vitamin E 1% (w/v) phospholipid emulsifier 1 g water to 100 gtotal

EXAMPLE 4 Liposome Composition

[0217] The following composition is prepared: LA-PLUS 100 mgdipalmitoylphosphatidyl- 900 mg choline (DPPC) buffered saline  10 ml

EXAMPLE 5 Cream Composition

[0218] The following composition is prepared: white paraffin 30 gvitamin E acetate  1 g LA-PLUS  1 g emulsifying wax 10 g water to 100 gtotal

EXAMPLE 6 Viability of HT4 Cells Following Exposure to Glutamate

[0219] The effects of compositions according to the invention on theviability of HT-4 cells treated with glutamate were determined. SevenHT-4 cell cultures were prepared and treated as follows: 1 Control (noglutamate) 2 Treatment with 10 mM glutamate for 12 hours 3 Treatmentwith 1 μM sodium selenite for 24 hours, followed by treatment with 10 mMglutamate for 12 hours 4 Treatment with 1 μM sodium selenite for 24hours, followed by treatment with 10 mM glutamate and 10 μM lipoic acidfor 12 hours 5 Treatment with 1 μM sodium selenite for 24 hours,followed by treatment with 10 mM glutamate and 10 μM LA-PLUS for 12hours 6 Twenty-four hours cell growth without sodium selenite followedby treatment with 10 mM glutamate and 10 μM lipoic acid for 12 hours 7Twenty-four hours cell growth without sodium selenite followed bytreatment with 10 mM glutamate and 10 μM LA-PLUS for 12 hours

[0220] Results are shown in FIG. 9. Enhanced cell viability was observedin the culture treated with combinations of LA-PLUS and sodium selenite(culture 5), and to a lesser extent, the culture treated with lipoicacid and sodium selenite (culture 4).

What is claimed is:
 1. A compound having the formula I:

wherein R¹ and R² independently denote a methylene, ethylene orunbranched or branched C₃₋₁₆ alkylene, alkenylene or alkynylene groupwhich is unsubstituted or substituted with one or more halogen, hydroxylor amine groups, wherein in said unbranched or branched C₃₋₁₆ alkylene,alkenylene or alkynylene group an internal alkylene carbon atom in thecarbon backbone thereof can be replaced by an oxygen atom, R³ and R⁴ (i)independently denote (a) hydrogen, (b) a methyl, ethyl, vinyl orunbranched or branched C₃₋₁₆ alkyl, alkenyl or alkynyl group which isunsubstituted or substituted with one or more halogen, hydroxyl or aminegroups, wherein in said unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group an internal alkylene carbon atom in the carbon backbonethereof can be replaced by an oxygen atom, (c) a cycloalkyl,alkylcycloalkyl, alkenylcycloalkyl or alkynylcycloalkyl group having 5to 16 carbon atoms which is unsubstituted or substituted with one ormore halogen, hydroxyl or amine groups, or (d) an aryl, alkaryl,aralkyl, alkenylaryl, aralkenyl, alkynylaryl or aralkynyl group having 6to 16 carbon atoms which is unsubstituted or substituted with one ormore halogen, hydroxyl or amine groups, or (ii) jointly with thenitrogen atom form a cyclic or aromatic amine which is unsubstituted orsubstituted with one or more alkyl, alkenyl, alkynyl, halogen, hydroxylor amine groups, X denotes O, S, —NH— or —NR⁵—, and R⁵ denotes methyl,ethyl, or unbranched or branched C₃₋₁₆ alkyl, or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1 wherein saidcompound is in equilibrium with a protonated form thereof atphysiological pH.
 3. The compound of claim 1 wherein X=—NH—.
 4. Thecompound of claim 3 wherein R¹ is —(CH₂)₄— and R² is —(CH₂)₂—
 5. Thecompound of claim 4 wherein R³ and R⁴ are methyl groups.
 6. The compoundof claim 4 wherein R³ and R⁴ form an unsubstituted or substitutedpyridine or imidazole group together with said nitrogen atom.
 7. Acompound having the formula II:

wherein L¹ and L² independently denote (i) a methylene group or a C₆₋₁₀arylene group which is unsubstituted or substituted with a halogen,hydroxyl, amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group or ii) a linking group having a carbon backbone thatincludes 2 to 16 carbon atoms, wherein a carbon atom in said carbonbackbone can be replaced by an oxygen atom, an unsubstituted orsubstituted amine group, a sulfur atom, an unsubstituted or substitutedC₆₋₁₀ aryl group or a combination thereof, Y denotes an ester,thioester, urethane or unsubstituted or alkyl-substituted amide linkage,and A denotes a group containing a nitrogen atom that can be protonatedor a pharmaceutically acceptable salt thereof.
 8. A compound having theformula III:

wherein W¹, W² (i) jointly denote —S—S—, —S(O)—S— or —S—S(O)—, or (ii)individually denote a group including an oxidized sulfur atom, L³denotes (i) a single bond, (ii) a methylene group or a C₆₋₁₀ arylenegroup which is unsubstituted or substituted with a halogen, hydroxyl,amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl or alkynyl group or(ii) a linking group having a carbon backbone that includes 2 to 16carbon atoms, wherein a carbon atom in said carbon backbone can bereplaced by an oxygen atom, an unsubstituted or substituted amine group,a sulfur atom, an unsubstituted or substituted C₆₋₁₀ aryl group or acombination thereof. Y denotes an ester, thioester, urethane orunsubstituted or alkyl-substituted amide linkage, n denotes 0 or 1, andZ denotes a group containing a nitrogen atom that is in equilibrium witha protonated form thereof and increases the intracellular retention timeof said compound with respect to the analogous compound having acarboxyl group, or a pharmaceutically acceptable salt thereof.
 9. Amethod of making a compound having the formula I

wherein R¹ and R² independently denote a methylene, ethylene orunbranched or branched C₃₋₁₆ alkylene, alkenylene or alkynylene groupwhich is unsubstituted or substituted with one or more halogen, hydroxylor amine groups, wherein in said unbranched or branched C₃₋₁₆ alkylene,alkenylene or alkynylene group an internal alkylene carbon atom in thecarbon backbone thereof can be replaced by an oxygen atom, R³ and R⁴ (i)independently denote (a) hydrogen, (b) a methyl, ethyl, vinyl orunbranched or branched C₃₋₁₆ alkyl, alkenyl or alkynyl group which isunsubstituted or substituted with one or more halogen, hydroxyl or aminegroups, wherein in said unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group an internal alkylene carbon atom in the carbon backbonethereof can be replaced by an oxygen atom, (c) a cycloalkyl,alkylcycloalkyl, alkenylcycloalkyl or alkynylcycloalkyl group having 5to 16 carbon atoms which is unsubstituted or substituted with one ormore halogen, hydroxyl or amine groups, or (d) an aryl, alkaryl,aralkyl, alkenylaryl, aralkenyl, alkynylaryl or aralkynyl group having 6to 16 carbon atoms which is unsubstituted or substituted with one ormore halogen, hydroxyl or amine groups, or (ii) jointly with thenitrogen atom form a cyclic or aromatic amine which is unsubstituted orsubstituted with one or more alkyl, alkenyl, alkynyl, halogen, hydroxylor amine groups, X denotes O, S, —NH— or —NR⁵—, and R⁵ denotes methyl,ethyl, or unbranched or branched C₃₋₁₆ alkyl, said method including thestep of reacting a compound having the formula IV

with a compound having the formula V R⁶—R²—NR³R⁴   V wherein R⁶ denotesOH, SH, NH₂ or NHR⁵.
 10. The method of claim 9 wherein said compoundhaving the formula IV is lipoic acid.
 11. The method of claim 10 whereinsaid compound having the formula V is selected from the group consistingof ethylenediamine, N,N-dimethylethylenediamine, ethanolamine,N,N-dimethylethanolamine, 2-pyridylethylamine and2-imidazolylethylamine.
 12. A method of making a compound having theformula II

wherein L¹ and L² independently denote (i) a methylene group or a C₆₋₁₀arylene group which is unsubstituted or substituted with a halogen,hydroxyl, amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl oralkynyl group or (ii) a linking group having a carbon backbone thatincludes 2 to 16 carbon atoms, wherein a carbon atom in said carbonbackbone can be replaced by an oxygen atom, an unsubstituted orsubstituted amine group, a sulfur atom, an unsubstituted or substitutedC₆₋₁₀ aryl group or a combination thereof, Y denotes an ester,thioester, urethane or unsubstituted or alkyl-substituted amide linkage,and A denotes a group containing a nitrogen atom that is in equilibriumwith a protonated form thereof, said method including the step ofreacting a compound having the formula VI

with a compound having the formula VII Y²-L²-A   VII wherein Y¹ and Y²denote groups which react to form an ester, thioester, urethane orunsubstituted or substituted amide linkage.
 13. The method of claim 12wherein one of said Y¹ and Y² groups is a —COOH group and the other ofsaid Y¹ and Y² groups is selected from the group consisting of —NH₂,—NHR⁵, —OH and —SH, wherein R⁵ denotes methyl, ethyl, or unbranched orbranched C₃₋₁₆ alkyl.
 14. A method of making a compound having theformula III

wherein W¹, W² (i) jointly denote —S—S—, —S(O)—S— or —S—S(O)—, or (ii)individually denote a group including an oxidized sulfur atom, L³denotes (i) a single bond, (ii) a methylene group or a C₆₋₁₀ arylenegroup which is unsubstituted or substituted with a halogen, hydroxyl,amine or unbranched or branched C₃₋₁₆ alkyl, alkenyl or alkynyl group or(ii) a linking group having a carbon backbone that includes 2 to 16carbon atoms, wherein a carbon atom in said carbon backbone can bereplaced by an oxygen atom, an unsubstituted or substituted amine group,a sulfur atom, an unsubstituted or substituted C₆₋₁₀ aryl group or acombination thereof, Y denotes an ester, thioester, urethane orunsubstituted or alkyl-substituted amine linkage, n denotes 0 or 1, andZ denotes a group containing a nitrogen atom that is in equilibrium witha protonated form thereof and increases the intracellular retention timeof said compound, said method including the step of reacting a compoundhaving the formula VI

with a compound having the formula VIII Y⁴   VIII to form a product,wherein Y³ denotes Y¹ or a group that reacts with Y⁴ to form an iminegroup or a guanidine group, Y⁴ denotes Y²-Z or a reagent that reactswith Y³to form an imine group or a guanidine group, and Y¹ and Y² denotegroups which react to form an ester, thioester, urethane orunsubstituted or substituted amide linkage.
 15. The method of claim 14wherein W¹, W² jointly denote —S(O)—S— or —S—S(O)—, or individuallydenote a group including an oxidized sulfur atom, and wherein saidmethod further comprises the step of oxidizing said product.
 16. Apharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 17. A pharmaceutical compositioncomprising a compound of claim 7 and a pharmaceutically acceptablecarrier.
 18. A pharmaceutical composition comprising a compound of claim8 and a pharmaceutically acceptable carrier.
 19. The pharmaceuticalcomposition of claim 18 further comprising an additional antioxidant.20. The pharmaceutical composition of claim 19 wherein said additionalantioxidant is sodium selenite.
 21. A cosmetic preparation comprising acompound of claim 1 and a cosmetic carrier.
 22. A cosmetic preparationcomprising a compound of claim 7 and a cosmetic carrier.
 23. A cosmeticpreparation comprising a compound of claim 8 and a cosmetic carrier. 24.A method of treating a condition in a warm-blooded animal that involvesa reactive oxygen species or a redox mechanism, said method comprisingthe step of administering to a warm-blooded animal having said conditionan effective amount of a compound of claim 1 .
 25. The method of claim24 wherein said condition is a pathological condition selected from thegroup consisting of exercise-induced tissue damage and physicalperformance, diabetes, atherosclerosis, an autoimmune disease, adegenerative brain disorder, a neoplastic disease, cerebral ischemia, ahepatic disorder, trauma, cachexia and AIDS.
 26. The method of claim 24wherein said condition is a clinical condition in which apoptosis ornecrosis is implicated in pathogenesis.
 27. The method of claim 24wherein said condition is an oxidation process associated with aging.28. A method of treating a condition in a warm-blooded animal thatinvolves a reactive nitrogen species, said method comprising the step ofadministering to a warm-blooded animal having said condition aneffective amount of a compound of claim 1 .